International audienceMosaicism is an important feature of type-1 neurofibromatosis (NF1) on account of its impact upon both clinical manifestations and transmission risk. Using FISH and MLPA to screen 3500 NF1 patients, we identified 146 individuals harbouring gross NF1 deletions, 14 of whom (9.6%) displayed somatic mosaicism. The high rate of mosaicism in patients with NF1 deletions supports the postulated idea of a direct relationship between the high new mutation rate in this cancer predisposition syndrome and the frequency of mosaicism. Seven of the 14 mosaic NF1 deletions were type-2, whereas 4 were putatively type-1, and three were atypical. Two of the 4 probable type-1 deletions were confirmed as such by breakpoint-spanning PCR or SNP analysis. Both deletions were associated with a generalized manifestation of NF1. Independently, we identified a third patient with a mosaic type-1 NF1 deletion who exhibited segmental NF1. Together, these three cases constitute the first proven mosaic type-1 deletions so far reported. In two of these three mosaic type-1 deletions, the breakpoints were located within PRS1 and PRS2, previously identified as hotspots for non-allelic homologous recombination (NAHR) during meiosis. Hence, NAHR within PRS1 and PRS2 is not confined to meiosis but may also occur during postzygotic mitotic cell cycles

BackgroundGenomic disorders are caused by copy number changes that may exhibit recurrent breakpoints processed by nonallelic homologous recombination. However, region-specific disease-associated copy number changes have also been observed which exhibit non-recurrent breakpoints. The mechanisms underlying these non-recurrent copy number changes have not yet been fully elucidated.ResultsWe analyze large NF1 deletions with non-recurrent breakpoints as a model to investigate the full spectrum of causative mechanisms, and observe that they are mediated by various DNA double strand break repair mechanisms, as well as aberrant replication. Further, two of the 17 NF1 deletions with non-recurrent breakpoints, identified in unrelated patients, occur in association with the concomitant insertion of SINE/variable number of tandem repeats/Alu (SVA) retrotransposons at the deletion breakpoints. The respective breakpoints are refractory to analysis by standard breakpoint-spanning PCRs and are only identified by means of optimized PCR protocols designed to amplify across GC-rich sequences. The SVA elements are integrated within SUZ12P intron 8 in both patients, and were mediated by target-primed reverse transcription of SVA mRNA intermediates derived from retrotranspositionally active source elements. Both SVA insertions occurred during early postzygotic development and are uniquely associated with large deletions of 1 Mb and 867 kb, respectively, at the insertion sites.ConclusionsSince active SVA elements are abundant in the human genome and the retrotranspositional activity of many SVA source elements is high, SVA insertion-associated large genomic deletions encompassing many hundreds of kilobases could constitute a novel and as yet under-appreciated mechanism underlying large-scale copy number changes in the human genome.

Targeted next‐generation‐sequencing (NGS) panels have largely replaced Sanger sequencing in clinical diagnostics. They allow for the detection of copy‐number variations (CNVs) in addition to single‐nucleotide variants and small insertions/deletions. However, existing computational CNV detection methods have shortcomings regarding accuracy, quality control (QC), incidental findings, and user‐friendliness. We developed panelcn.MOPS, a novel pipeline for detecting CNVs in targeted NGS panel data. Using data from 180 samples, we compared panelcn.MOPS with five state‐of‐the‐art methods. With panelcn.MOPS leading the field, most methods achieved comparably high accuracy. panelcn.MOPS reliably detected CNVs ranging in size from part of a region of interest (ROI), to whole genes, which may comprise all ROIs investigated in a given sample. The latter is enabled by analyzing reads from all ROIs of the panel, but presenting results exclusively for user‐selected genes, thus avoiding incidental findings. Additionally, panelcn.MOPS offers QC criteria not only for samples, but also for individual ROIs within a sample, which increases the confidence in called CNVs. panelcn.MOPS is freely available both as R package and standalone software with graphical user interface that is easy to use for clinical geneticists without any programming experience. panelcn.MOPS combines high sensitivity and specificity with user‐friendliness rendering it highly suitable for routine clinical diagnostics.

Nonallelic homologous recombination (NAHR) is one of the major mechanisms underlying copy number variation in the human genome. Although several disease-associated meiotic NAHR breakpoints have been analyzed in great detail, hotspots for mitotic NAHR are not well characterized. Type-2 NF1 microdeletions, which are predominantly of postzygotic origin, constitute a highly informative model with which to investigate the features of mitotic NAHR. Here, a custom-designed MLPA- and PCR-based approach was used to identify 23 novel NAHR-mediated type-2 NF1 deletions. Breakpoint analysis of these 23 type-2 deletions, together with 17 NAHR-mediated type-2 deletions identified previously, revealed that the breakpoints are nonuniformly distributed within the paralogous SUZ12 and SUZ12P sequences. Further, the analysis of this large group of type-2 deletions revealed breakpoint recurrence within short segments (ranging in size from 57 to 253-bp) as well as the existence of a novel NAHR hotspot of 1.9-kb (termed PRS4). This hotspot harbored 20% (8/40) of the type-2 deletion breakpoints and contains the 253-bp recurrent breakpoint region BR6 in which four independent type-2 deletion breakpoints were identified. Our findings indicate that a combination of an open chromatin conformation and short non-B DNA-forming repeats may predispose to recurrent mitotic NAHR events between SUZ12 and its pseudogene.

Nonallelic homologous recombination (NAHR) is responsible for the recurrent rearrangements that give rise to genomic disorders. Although meiotic NAHR has been investigated in multiple contexts, much less is known about mitotic NAHR despite its importance for tumorigenesis. Because type-2 NF1 microdeletions frequently result from mitotic NAHR, they represent a good model in which to investigate the features of mitotic NAHR. We have used microsatellite analysis and SNP arrays to distinguish between the various alternative recombinational possibilities, thereby ascertaining that 17 of 18 type-2 NF1 deletions, with breakpoints in the SUZ12 gene and its highly homologous pseudogene, originated via intrachromosomal recombination. This high proportion of intrachromosomal NAHR causing somatic type-2 NF1 deletions contrasts with the interchromosomal origin of germline type-1 NF1 microdeletions, whose breakpoints are located within the NF1-REPs (low-copy repeats located adjacent to the SUZ12 sequences). Further, meiotic NAHR causing type-1 NF1 deletions occurs within recombination hotspots characterized by high GC-content and DNA duplex stability, whereas the type-2 breakpoints associated with the mitotic NAHR events investigated here do not cluster within hotspots and are located within regions of significantly lower GC-content and DNA stability. Our findings therefore point to fundamental mechanistic differences between the determinants of mitotic and meiotic NAHR.